Clinical Science (1992) 83, 277-280 (Printed in Great Britain)
277
Rapid Communication
Pancreatic secretion of lysosomal enzymes stimulated by
intraduodenal instillation of a liquid meal in rabbits
T. HIRANO, T. MANABE, A.
K. SALUJA* and M. L. STEER*
First Department of Surgery. Faculty of Medicine, Kyoto University, Kyoto, japan, and
*Department of Surgery. Beth Israel Hospital and Harvard Medical School, Boston,
Massachusetts, U.S.A.
(Received 9 June 1992; accepted 3 July 1992)
1. Studies have been performed to determine the
effect of intraduodenal food on pancreatic secretion
of lysosomal enzymes.
2. Intraduodenal instillation of a liquid meal (3g/kg
body weight; 15.3% protein, 19.7% fat, 59.7% carbohydrate) caused significant increases in pancreatic
juice volume and pancreatic secretion of amylase and
protein compared with basal values for 2 h after
instillation in anaesthetized rabbits.
3. Intraduodenal instillation of a liquid meal also
caused significant increases in pancreatic secretion of
three lysosomal enzymes (cathepsin B, N-fi-acetylgalactosaminidase and N-acetyl-b-glucosaminidase)
compared with basal values for 2 h after instillation.
4. In addition, there were significant correlations
between cathepsin B secretion and amylase secretion
(r=0.7764, P< 0.001) and between cathepsin B secretion and protein secretion (r=0.6216, P< 0.001), both
in basal conditions and in response to the liquid meal.
5. These results are evidence for the localization of
lysosomal enzymes in the secretory granules-zymogen granules in normal acinar cells, and also indicate
that the pancreatic secretion of lysosomal enzymes is
gut-hormone-regulated.
responsible for the pancreatic secretion of lysosomal
enzymes in normal physiological states, such as in
response to food intake. In this study, we report the
stimulation by food intake of lysosomal enzyme
secretion from the exocrine pancreas.
METH6DS
Animals
Six New Zealand White rabbits, weighing about
2.5 kg, were obtained from Shizuoka Experimental
Animal Supply (Shizuoka, Japan). They were kept
in light-dark cycle regulated (light: 05.0G17.00
hours) and air-conditioned (23 rt 3°C) animal
quarters in our institute. They were given free access
to tap water and food (Oriental Rodent Chow,
Tokyo, Japan), and were allowed to acclimatize to
the standard laboratory conditions for at least 4
days. The rabbits were maintained throughout the
study in accordance with the guidelines of the
Committee on Animal Care of Kyoto University.
Chemicals
INTRODUCTION
There have been several reports on the secretory
profiles of lysosomal enzymes in cell lines [l-31, and
it has been suggested that there may be a physiological role for lysosomal enzymes in biological
fluids [4]. Most of these studies have been performed using non-polarized cell types and the
results of these studies have indicated that, under
most conditions, lysosomal enzymes are secreted in
a constitutive manner [5, 61.
However, little is known about the mechanisms
Isocal Meal (15.3% protein, 19.7% fat, 59.7%
carbohydrate) was purchased from Mead Johnson
(Evansville, IN, U.S.A.). Benzyloxycarbonyl-arginylarginine-P-naphthylamide was obtained from
AG
(Budendorf,
Bachem
Feinchemikalien
Switzerland) and P-naphthylamine was purchased
from Sigma Chemical Co. (St Louis, MO, U.S.A.).
4-Methylumbelliferyl substrates for N-acetyl-Pglucosaminidase and N-P-acetylgalactosaminidase
were purchased from Sigma Chemical Co. All other
reagents were of the highest purity commercially
available.
Key words: N-P-acetylgalaaoraminidase, N-acetyl-8-glucosaminidase, cathepsin 6, lysosomal enzymes, pancreatic juice.
Correspondence: Dr Teuuya Hirano, First Department of Surgery, Faculty of Medicine, Kyoto University, 62-10 Shirhigatani-Teranomaecho, Sakyoku, Kyoto 606, Japan.
270
T. Hirano et al.
Animal preparation
Assays
After a 16 h fast, rabbits were anaesthetized with
an intravenous injection of pentobarbital (30mg/kg
initially, supplemented by periodic doses of lOmg/kg)
and a cannula (PE-50; Clay Adams, Parsippany, NJ,
U.S.A.) was introduced into the inferior vena cava
via the right femoral vein for the venous line. After
laparotomy, the pylorus was ligated and a drainage
gastrostomy cannula (PE-70) was positioned. The
pancreatic duct was cannulated (PE-50) extraduodenally, adjacent to the duodenum, and another
cannula (PE-50) was positioned in the descending
portion of the duodenum just distal to the pylorus. 1
After placement and exteriorization of various '
catheters, the abdominal wound was closed. The
core temperature of the rabbits was maintained
using a heating pad and overhead lamps, and
during the experiments rabbits were infused with
intravenous heparinized saline (150 mmol/l NaCI) at
a rate of 1.58ml/h.
After a 30min stabilization, the first l'h pancreatic juice fraction was collected as a basal value
(B) and then liquid meal (Isocal Meal, 3g/kg body
weight in 15ml of water) was instilled into the
duodenum through the duodenostomy catheter over
15min using an infusion pump. After a basal sample
collection, three further 1 h collections of pancreatic
juice (D1, D, and D3) were obtained during and
after the instillation of the liquid meal.
For each fraction, protein concentration and the
activities of the enzymes amylase, cathepsin By
N-P-acetylgalactosaminidase
and
N-acetyl-Pglucosaminidase were measured. Protein secretion
was expressed in mgh-'kg-' and all enzyme secretions were expressed in units h-lkg-'.
Amylase activity was measured by the method of
Irie et al. [7], with blue starch (Shionogi Amylase
A-Test; Shionogi, Osaka, Japan) as the substrate.
One unit of activity was defined as that which
liberated l m g of maltose from the substrate/min at
30°C. Cathepsin B activity was measured by the
with
method of McDonald & Ellis [S]
benzyloxycarbonyl-arginyl-arginine-P-naphthylamide
as the substrate, and one unit of activity was defied
as that which liberated 1 nmol of P-naphthylaminel
min at 37°C. The activities of N-/I-acetylgalactosaminidase (EC 3.2.1.53) and N-acetyl-Pglucosaminidase (EC 3.2.1.30) were measured by the
method of Peters et al. [9]. One unit of these two
enzymes was defined as that which hydroIysed
1nmol of substrate/min. Protein concentration was
measured by the method of Lowry et al. [lo] with
BSA as the standard.
Statistical analysis
The results are reported as means )SEM for n
determinations. The significance of changes was
evaluated by analysis of variance and the Tukey
procedure, and a P value of less than 0.05 was
considered to be significant.
RESULTS
Intraduodenal instillation of a liquid meal
resulted in the stimulation of pancreatic juice secretion (By 0.124+0.010; D,, 0.273+0.018; D,,
0.230 )0.014; D,, 0.147 & 0.012 ml h - kg - l), and
this increase (two- to three-fold compared with the
*
T
ir*
**
T
B
D,
Dz D,
Fig. I. Secretion of pancreatic juice (a), amylase (b) and protein (c) from the rabbit exocrine pancreas stimulated by
intraduodenal instillation of a liquid meal. There were six rabbits in this experiment, and each rabbit produced one basal
fraction (B) amd three post-stimulation fractions (DI, Dz and D,) each collected over I h. Values are means +SEH. Statistical
significance: *P<O.Ol, **P<0.02, ***P<0.05 compared with the basal fraction (B).
Effect
of intraduodenal food on pancreatic secretion of lysosomal enzymes
279
Fig. 2. Secretion of cathepsin B (a), Nacetyl-Bglucoraminidase (b) and Nficetylgalactosaminidase (c) into rabbit
pancreatic juice stimulated by intraduodenal instillation of a liquid meal. The rabbits were the same as those in Fig. I, and
fractions were collected in the same manner as described in the legend t o Fig. I. Values are means +$EM. Statistical significance:
*P<O.OI, **P<0.02, ***P<0.05 compared with the basal fraction (6).
3.3k0.4; D,, 1.7A0.3 units h-lkg', Figs. 2b and
2c).
In addition, the secretion of cathepsin B in response to the meal correlated closely with that of
amylase ( r = 0.7764, P 0.001) and protein
(r=0.6216, P<O.OOl, Fig. 3).
-=
DISCUSSION
I
2
4
6
8
Cathepsin B secretion (units h - l k g - l )
I
2
4
6
8
Cathepsin B secretion (units h - l k g - l )
Fig. 3. Relationships between cathepsin B secretion and amylase
secretion (a), and between cathepsin B secretion and protein
secretion (b), in rabbit pancreatic juice (basal fraction and
liquidmeal-stimulated fractions combined). (a) y = 34x+ 113,
r=0.7764, P<O.OOl. (b) y = l . I x + S . l , r=0.6216, P<O.OOl.
basal volume) continued for almost 2 h in anaesthetized rabbits (Fig. la). Furthermore, intraduodenal
instillation of food stimulated the secretion of amylase (B, 147i-16; D,, 343+30, D,, 262i-18; D,,
171k16 units h-lkg-', Fig. lb) and protein (B,
5.4k0.8; D,, 13.1k2.0; D,,
10.0+1.2; D,,
7.3+1.6rngh-'kg-',
Fig. lc).
Intraduodenal instillation of a liquid meal caused
the stimulation of cathepsin B secretion into pancreatic juice (B, 1.8f0.6, D,, 5.8f1.4, D,, 4.0k0.8;
D,, 2.3k0.6 units h-' kg-l, Fig. 24. Similar stimulation of the other lysosomal enzymes occurred over
the 2 h after duodenal food instillation (N-acetyl-Pglucosaminidase: B; 2.0k0.2; D1, 6.3+ 1.1; D,, 5.8k
0.5; D,, 2.6k0.5 units h-'kg;
N-P-acetylgalactosaminidase: B, 1.3 f0.4; D,, 4.2 k0.5; D,,
Protein secretion by pancreatic acinar cells is a
complex process, which, depending on the protein
involved, may utilize either regulated or constitutive
pathways, and, in these polarized cells, be confined
to either the apical (i.e. luminal) or basolateral cell
surfaces. Digestive enzymes, for example, appear to
be discharged only at the apical surface and to be
primarily secreted in a regulated fashion, whereas
proteins, such as those comprising the basement
membrane, are believed to be constitutively secreted
at the basolateral cell surface [ 5 , 61.
Lysosomal hydrolases comprise only a small fraction of acinar cell proteins and only a small portion
of the lysosomal enzyme mass is secreted. Those
lysosomal enzymes which are not secreted are
diverted from the secretory pathway and transported to the acidic pre-lysosomal (endosomal)
compartment. Sorting of lysosomal hydrolases from
secretory enzymes is believed to occur in the transGolgi network where the mannose-6-phosphorylated
lysosomal hydrolases are captured by mannose-6phosphate-specific receptors and packed in transport vesicles [ l l , 121.
Secretion of lysosomal hydrolases has been
observed to occur in many types of cells and it is
generally believed that lysosomal enzyme secretion
is a constitutive process [S, 61. We have studied the
secretion of lysosomal hydrolases into the pancreatic juice and, in this way, confined our observations to changes reflecting discharge across the
apical surface of exocrine pancreatic cells.
280
T. Hirano et al.
There is also a close correlation between cathepsin B secretion and amylase secretion after the
instillation of nutrients into the duodenum, a response which is presumably mediated by endogenously released hormones and neurotransmitters.
These observations strongly suggest that the stimulated secretion of lysosomal enzymes into the pancreatic juice reflects a response of acinar cells and
that the secreted digestive enzymes and lysosomal
enzymes are discharged from the same pre-secretory
compartment, i.e. zymogen granules. This is supported by reports of the co-localization of lysosomal
enzymes with digestive enzymes in normal acinar
cells [13, 141.
An alternative interpretation, which can not be
excluded but which, because of its complexity, seems
to be less attractive, would be that lysosomal and
digestive enzymes are discharged at the apical cell
surface from separate compartments, each of which
is regulated by a secretagogue. In either case, however, it is clear that apical discharge of lysosomal
hydrolases from rabbit acinar cells is primarily a
regulated process which is closely correlated with
the secretion of digestive zymogens.
This co-localization of digestive enzymes and
lysosomal hydrolases in zymogen granules could
result from several potential mechanisms. Lysosoma1 enzymes might be trapped in the bulk flow of
digestive enzymes and thus inadvertently packaged
in condensing vacuoles. Failure of mannose 6phosphate receptors to capture all of the lysosomal
enzymes could indicate that there are inadequate
numbers of receptors, or that, for steric or other
reasons, binding of enzymes to receptors is constrained. Finally, it is possible that sorting during
transit through the trans-Golgi network is complete,
but subsequent to sorting lysosomal enzymecontaining organelles fuse with zymogen-containing
structures and lysosomal hydrolases are re-inserted
into the digestive enzyme secretory pathway. Studies
employing techniques of immunolocalization will be
needed to evaluate these possibilities. Further
studies should also address the possibility that
lysosomal hydrolases may also be discharged from
the basolateral surface of acinar cells via either
constitutive or regulated mechanisms, since such
discharge would have gone undetected during the
present study.
Since the lysosomal hydrolase, cathepsin B, can
activate trypsinogen [lS, 161, and trypsin can activate many other digestive enzymes, this colocalization of lysosomal hydrolases and digestive
enzymes in zymogen granules both in normal acinar
cells and in normal pancreatic juice might shed light
on the pathophysiology of pancreatitis, and also
indicates the need to investigate the possible physiological roles of lysosomal enzymes in pancreatic
juice.
ACKN 0W LEDGMENTS
This work was supported by a grant from the
Ministry of Education, Science and Culture of
Japan (Scientific Research B-62480282) and a grant
from the National Institutes of Health (DK-31396
and DK-31394). We thank Ms Yoko Manabe and
Ms Kimiko Hirano for typing the manuscript.
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